Integrated analysis of transcriptomics and metabolomics of garden asparagus (Asparagus officinalis L.) under drought stress.

BACKGROUND: Drought is a leading environmental factor affecting plant growth. To explore the drought tolerance mechanism of asparagus, this study analyzed the responses of two asparagus varieties, namely, 'Jilv3' (drought tolerant) and 'Pacific Early' (drought sensitive), to drought stress using metabolomics and transcriptomics. RESULTS: In total, 2,567 and 7,187 differentially expressed genes (DEGs) were identified in 'Pacific Early' and 'Jilv3', respectively, by comparing the transcriptome expression patterns between the normal watering treatment and the drought stress treatment. These DEGs were significantly enriched in the amino acid biosynthesis, carbon metabolism, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. In 'Jilv3', DEGs were also enriched in the following energy metabolism-related pathways: citrate cycle (TCA cycle), glycolysis/gluconeogenesis, and pyruvate metabolism. This study also identified 112 and 254 differentially accumulated metabolites (DAMs) in 'Pacific Early' and 'Jilv3' under drought stress compared with normal watering, respectively. The amino acid, flavonoid, organic acid, and soluble sugar contents were more significantly enhanced in 'Jilv3' than in 'Pacific Early'. According to the metabolome and transcriptome analysis, in 'Jilv3', the energy supply of the TCA cycle was improved, and flavonoid biosynthesis increased. As a result, its adaptability to drought stress improved. CONCLUSIONS: These findings help to better reveal the molecular mechanism underlying how asparagus responds to drought stress and improve researchers' ability to screen drought-tolerant asparagus varieties as well as breed new varieties.

Physiological and transcriptome analysis of changes in endogenous hormone and sugar content during the formation of tender asparagus stems.

Asparagus is a nutritionally dense stem vegetable whose growth and development are correlated with its quality and yield. To investigate the dynamic changes and underlying mechanisms during the elongation and growth process of asparagus stems, we documented the growth pattern of asparagus and selected stem segments from four consecutive elongation stages using physiological and transcriptome analyses. Notably, the growth rate of asparagus accelerated at a length of 25 cm. A significant decrease in the concentration of sucrose, fructose, glucose, and additional sugars was observed in the elongation region of tender stems. Conversely, the levels of auxin and gibberellins(GAs) were elevated along with increased activity of enzymes involved in sucrose degradation. A significant positive correlation existed between auxin, GAs, and enzymes involved in sucrose degradation. The ABA content gradually increased with stem elongation. The tissue section showed that cell elongation is an inherent manifestation of stem elongation. The differential genes screened by transcriptome analysis were enriched in pathways such as starch and sucrose metabolism, phytohormone synthesis metabolism, and signal transduction. The expression levels of genes such as ARF, GA20ox, NCED, PIF4, and otherswere upregulated during stem elongation, while DAO, GA2ox, and other genes were downregulated. The gene expression level was consistent with changes in hormone content and influenced the cell length elongation. Additionally, the expression results of RT-qPCR were consistent with RNA-seq. The observed variations in gene expression levels, endogenous hormones and sugar changes during the elongation and growth of asparagus tender stems offer valuable insights for future investigations into the molecular mechanisms of asparagus stem growth and development and provide a theoretical foundation for cultivation and production practices.

Screening and Preliminary Identification of Asparagus officinalis Varieties under Low-Temperature Stress.

To meet the large demand for Asparagus officinalis in the spring market and improve the economic benefits of cultivating asparagus, we explored the molecular mechanism underlying the response of A. officinalis to low temperature. First, "Fengdao No. 1" was screened out under low-temperature treatment. Then, the transcriptome sequencing and hormone detection of "Fengdao No. 1" and "Grande" (control) were performed. Transcriptome sequencing resulted in screening out key candidate genes, while hormone analysis indicated that ABA was important for the response to low temperature. The combined analysis indicated that the AoMYB56 gene may regulate ABA in A. officinalis under low temperature. And the phylogenetic tree was constructed, and subcellular localisation was performed. From these results, we speculated that the AoMYB56 gene may regulate ABA in A. officinalis. The results of this research provide a theoretical basis for the further exploration of low-temperature response in A. officinalis.

Genome-wide identification and comprehensive analysis heat shock transcription factor (Hsf) members in asparagus (Asparagus officinalis) at the seeding stage under abiotic stresses.

Heat shock transcription factors (Hsf) are pivotal as essential transcription factors. They function as direct transcriptional activators of genes regulated by thermal stress and are closely associated with various abiotic stresses. Asparagus (Asparagus officinalis) is a vegetable of considerable economic and nutritional significance, abundant in essential vitamins, minerals, and dietary fiber. Nevertheless, asparagus is sensitive to environmental stresses, and specific abiotic stresses harm its yield and quality. In this context, Hsf members have been discerned through the reference genome, and a comprehensive analysis encompassing physical and chemical attributes, evolutionary aspects, motifs, gene structure, cis-acting elements, collinearity, and expression patterns under abiotic stresses has been conducted. The findings identified 18 members, categorized into five distinct subgroups. Members within each subgroup exhibited analogous motifs, gene structures, and cis-acting elements. Collinearity analysis unveiled a noteworthy pattern, revealing that Hsf members within asparagus shared one, two, and three pairs with counterparts in Arabidopsis, Oryza sativa, and Glycine max, respectively.Furthermore, members displayed tissue-specific expression during the seedling stage, with roots emerging as viable target tissue. Notably, the expression levels of certain members underwent modification under the influence of abiotic stresses. This study establishes a foundational framework for understanding Hsf members and offers valuable insights into the potential application of molecular breeding in the context of asparagus cultivation.

Comparative and phylogenetic analysis of Asparagus meioclados Levl. and Asparagus munitus Wang et S. C. Chen plastomes and utility of plastomes mutational hotspots.

Tiandong is a vital traditional Chinese herbal medicine. It is derived from the tuber root of the Asparagus cochinchinensis according to the Pharmacopoeia of the people's republic of China (2020 Edition). On account of the similar morphology, Asparagus meioclados and Asparagus munitus were used as Tian-Dong in southwest China. Chloroplast (cp) genomes are highly active genetic components of plants and play an extremely important role in improving the efficiency of the identification of plant species. To differentiate the medicinal plants belonging to the genus Asparagus, we sequenced and analyzed the complete plastomes (plastid genomes) of A. meioclados and A. munitus and obtained two plastomes whose length changed to 156,515 bp and 156,381 bp, respectively. A total of 111 unique genes have been detected in plastome, which included 78 protein-coding genes, 29 tRNA genes and 4 rRNA genes. In plastomes of A. meioclados and A. munitus, 14,685 and 14,987 codons were detected, among which 9942 and 10,207 had the relative synonymous codon usage (RSCU) values higher than 1, respectively. A. meioclados and A. munitus have 26 SSRs patterns, among which A. meioclados was 25 and A. munitus 21. The average Ka/Ks value was 0.36, and positive selection was detected in genes of the photosynthetic system (ndhF and rbcL) in Asparagus species. To perform the comparative analysis of plastomes, the two newly sequenced plastomes of the A. meioclados and A. munitus species were compared with that of A. cochinchinensis, and 12 hotspots, including 5 coding regions and 7 inter-genomic regions, were identified. Based on the whole plastome of Asparagus, 2 divergent hotspots (accD and rpl32-trnL-UAG) and 1 international barcode fragment (rbcL) were screened, which may be used as particular molecular markers for the identification of Asparagus species. In addition, we determined the phylogenetic relationship between A. meioclados and A. munitus in the genus Asparagus. This study enriches our knowledge of the molecular evolutionary relationships of the Asparagus genus and provides treasured data records for species identification, molecular breeding, and evolutionary analysis of this genus.

Steroidal saponin profiles and their key genes for synthesis and regulation in Asparagus officinalis L. by joint analysis of metabolomics and transcriptomics.

BACKGROUND: Asparagus officinalis L. is a worldwide cultivated vegetable enrichened in both nutrient and steroidal saponins with multiple pharmacological activities. The upstream biosynthetic pathway of steroidal saponins (USSP) for cholesterol (CHOL) synthesis has been studied, while the downstream pathway of steroidal saponins (DSSP) starting from cholesterol and its regulation in asparagus remains unknown. RESULTS: Metabolomics, Illumina RNAseq, and PacBio IsoSeq strategies were applied to different organs of both cultivated green and purple asparagus to detect the steroidal metabolite profiles & contents and to screen their key genes for biosynthesis and regulation. The results showed that there is a total of 427 compounds, among which 18 steroids were detected with fluctuated concentrations in roots, spears and flowering twigs of two garden asparagus cultivars. The key genes of DSSP include; steroid-16-hydroxylase (S16H), steroid-22-hydroxylase (S22H) and steroid-22-oxidase-16-hydroxylase (S22O-16H), steroid-26-hydroxylase (S26H), steroid-3-ß-glycosyltransferase (S3ßGT) and furostanol glycoside 26-O-beta-glucosidases (F26GHs) which were correlated with the contents of major steroidal saponins were screened, and the transcriptional factors (TFs) co-expressing with the resulted from synthetic key genes, including zinc fingers (ZFs), MYBs and WRKYs family genes were also screened. CONCLUSIONS: Based on the detected steroidal chemical structures, profiles and contents which correlated to the expressions of screened synthetic and TFs genes, the full steroidal saponin synthetic pathway (SSP) of asparagus, including its key regulation networks was proposed for the first time.

AoSAP8-P encoding A20 and/or AN1 type zinc finger protein in asparagus officinalis L. Improving stress tolerance in transgenic Nicotiana sylvestris.

The full length CDS of an A20 and AN1 type zinc finger gene (named AoSAP8-P), located nearby the male specific Y chromosome (MSY) region of Asparagus officinalis (garden asparagus) was amplified by RT-PCR from purple passion. This gene, predicted as the stress associated protein (SAPs) gene families, encodes 172 amino acids with multiple cis elements including light, stress response box, MYB and ERF binding sites on its promoter. To analyze its function, the gene expression of different organs in different asparagus gender were analyzed and the overexpressed transgenic Nicotiana sylvestris lines were generated. The results showed the gene was highly expressed in both flower and root of male garden asparagus; the germination rate of seeds of the T2 transgenic lines (T2-5-4 and T2-7-1) under the stress conditions of 125 mM NaCl and 150 mM mannitol were significantly higher than the wild type (WT) respectively. When the potted T2-5-4, T2-7-1 lines and WT were subjected to drought stress for 24 days and the leaf discs immerged into 20 % PEG6000 and 300 mM NaCl solution for 48 h respectively, the T2-5-4 and T2-7-1 with AoSAP8-P expression showed stronger drought, salt and osmotic stress tolerance. When compared, the effects of AoSAP8-P overexpression on productive development showed that the flowering time of transgenic lines, were âˆ¼ 9 day earlier with larger but fewer pollens than its WT counterparts. However, there were no significant differences in anthers, stigmas and pollen viability between the transgenic lines and WT. Our results suggested that, the AoSAP8-P gene plays a role in improving the stress resistance and shortening seeds generation time for perianal survival during the growth and development of garden asparagus.

A Joint Transcriptomic and Metabolomic Analysis Reveals the Regulation of Shading on Lignin Biosynthesis in Asparagus.

Asparagus belongs to the Liliaceae family and has important economic and pharmacological value. Lignin plays a crucial role in cell wall structural integrity, stem strength, water transport, mechanical support and plant resistance to pathogens. In this study, various biological methods were used to study the mechanism of shading on the asparagus lignin accumulation pathway. The physiological results showed that shading significantly reduced stem diameter and cell wall lignin content. Microstructure observation showed that shading reduced the number of vascular bundles and xylem area, resulting in decreased lignin content, and thus reducing the lignification of asparagus. Cinnamic acid, caffeic acid, ferulic acid and sinapyl alcohol are crucial intermediate metabolites in the process of lignin synthesis. Metabolomic profiling showed that shading significantly reduced the contents of cinnamic acid, caffeic acid, ferulic acid and sinapyl alcohol. Transcriptome profiling identified 37 differentially expressed genes related to lignin, including PAL, C4H, 4CL, CAD, CCR, POD, CCoAOMT, and F5H related enzyme activity regulation genes. The expression levels of POD, CCoAOMT, and CCR genes were significantly decreased under shading treatment, while the expression levels of CAD and F5H genes exhibited no significant difference with increased shading. The downregulation of POD, CCoAOMT genes and the decrease in CCR gene expression levels inhibited the activities of the corresponding enzymes under shading treatment, resulting in decreased downstream content of caffeic acid, ferulic acid, sinaperol, chlorogenic acid and coniferin. A significant decrease in upstream cinnamic acid content was observed with shading, which also led to decreased downstream metabolites and reduced asparagus lignin content. In this study, transcriptomic and metabolomic analysis revealed the key regulatory genes and metabolites of asparagus lignin under shading treatment. This study provides a reference for further understanding the mechanism of lignin biosynthesis and the interaction of related genes.

Genome-Wide Identification and Analysis of the NAC Transcription Factor Gene Family in Garden Asparagus (Asparagus officinalis).

As a large plant-specific gene family, the NAC (NAM, ATAF1/2, and CUC2) transcription factor is related to plant growth, development, and response to abiotic stresses. Although the draft genome of garden asparagus (Asparagus officinalis) has been released, the genome-wide investigation of the NAC gene family is still unavailable. In this study, a total of 85 A. officinalis NAC genes were identified, and a comprehensive analysis of the gene family was performed, including physicochemical properties, phylogenetic relationship, chromosome localization, gene structure, conserved motifs, intron/exon, cis-acting elements, gene duplication, syntenic analysis, and differential gene expression analysis. The phylogenetic analysis demonstrated that there were 14 subgroups in both A. officinalis and Arabidopsis thaliana, and the genes with a similar gene structure and motif distribution were clustered in the same group. The cis-acting regulatory analysis of AoNAC genes indicated four types of cis-acting elements were present in the promoter regions, including light-responsive, hormone-responsive, plant-growth-and-development-related, and stress-responsive elements. The chromosomal localization analysis found that 81 NAC genes in A. officinalis were unevenly distributed on nine chromosomes, and the gene duplication analysis showed three pairs of tandem duplicated genes and five pairs of segmental duplications, suggesting that gene duplication is possibly associated with the amplification of the A. officinalis NAC gene family. The differential gene expression analysis revealed one and three AoNAC genes that were upregulated and downregulated under different types of salinity stress, respectively. This study provides insight into the evolution, diversity, and characterization of NAC genes in garden asparagus and will be helpful for future understanding of their biological roles and molecular mechanisms in plants.

Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop 'Myers', and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics.

Asparagus species are widely used for medicinal, horticultural, and culinary purposes. Complete chloroplast DNA (cpDNA) genomes of three Asparagus specimens collected in Hong Kong-A. aethiopicus, A. densiflorus 'Myers', and A. cochinchinensis-were de novo assembled using Illumina sequencing. Their sizes ranged from 157,069 to 157,319 bp, with a total guanine-cytosine content of 37.5%. Structurally, a large single copy (84,598-85,350 bp) and a small single copy (18,677-18,685 bp) were separated by a pair of inverted repeats (26,518-26,573 bp). In total, 136 genes were annotated for A. aethiopicus and A. densiflorus 'Myers'; these included 90 mRNA, 38 tRNA, and 8 rRNA genes. Further, 132 genes, including 87 mRNA, 37 tRNA, and 8 rRNA genes, were annotated for A. cochinchinensis. For comparative and phylogenetic analysis, we included NCBI data for four congenerics, A. setaceus, A. racemosus, A. schoberioides, and A. officinalis. The gene content, order, and genome structure were relatively conserved among the genomes studied. There were similarities in simple sequence repeats in terms of repeat type, sequence complementarity, and cpDNA partition distribution. A. densiflorus 'Myers' had distinctive long sequence repeats in terms of their quantity, type, and length-interval frequency. Divergence hotspots, with nucleotide diversity (Pi) ≥ 0.015, were identified in five genomic regions: accD-psaI, ccsA, trnS-trnG, ycf1, and ndhC-trnV. Here, we summarise the historical changes in the generic subdivision of Asparagus. Our phylogenetic analysis, which also elucidates the nomenclatural complexity of A. aethiopicus and A. densiflorus 'Myers', further supports their close phylogenetic relationship. The findings are consistent with prior generic subdivisions, except for the placement of A. racemosus, which requires further study. These de novo assembled cpDNA genomes contribute valuable genomic resources and help to elucidate Asparagus taxonomy.

Chromosome-scale haplotype-phased genome assemblies of the male and female lines of wild asparagus (Asparagus kiusianus), a dioecious plant species.

Asparagus kiusianus is a disease-resistant dioecious plant species and a wild relative of garden asparagus (Asparagus officinalis). To enhance A. kiusianus genomic resources, advance plant science, and facilitate asparagus breeding, we determined the genome sequences of the male and female lines of A. kiusianus. Genome sequence reads obtained with a linked-read technology were assembled into four haplotype-phased contig sequences (∼1.6 Gb each) for the male and female lines. The contig sequences were aligned onto the chromosome sequences of garden asparagus to construct pseudomolecule sequences. Approximately 55,000 potential protein-encoding genes were predicted in each genome assembly, and ∼70% of the genome sequence was annotated as repetitive. Comparative analysis of the genomes of the two species revealed structural and sequence variants between the two species as well as between the male and female lines of each species. Genes with high sequence similarity with the male-specific sex determinant gene in A. officinalis, MSE1/AoMYB35/AspTDF1, were presented in the genomes of the male line but absent from the female genome assemblies. Overall, the genome sequence assemblies, gene sequences, and structural and sequence variants determined in this study will reveal the genetic mechanisms underlying sexual differentiation in plants, and will accelerate disease-resistance breeding in garden asparagus.

Decreased expression of fructosyltransferase genes in asparagus roots may contribute to efficient fructan degradation during asparagus spear harvesting.

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases-sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris, were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears.

Comparative Metabolome and Transcriptome Analyses of Susceptible Asparagus officinalis and Resistant Wild A. kiusianus Reveal Insights into Stem Blight Disease Resistance.

Phomopsis asparagi is one of the most serious fungal pathogens, which causes stem blight disease in Asparagus officinalis (AO), adversely affecting its production worldwide. Recently, the development of novel asparagus varieties using wild Asparagus genetic resources with natural P. asparagi resistance has become a priority in Japan due to the lack of resistant commercial AO cultivars. In this study, comparative metabolome and transcriptome analyses of susceptible AO and resistant wild Asparagus kiusianus (AK) 24 and 48 h postinoculated (AOI_24 hpi, AOI_48 hpi, AKI_24 hpi and AKI_48 hpi, respectively) with P. asparagi were conducted to gain insights into metabolic and expression changes associated with AK species. Following infection, the resistant wild AK showed rapid metabolic changes with increased levels of flavonoids and steroidal saponins and decreased asparagusic acid glucose ester content, compared with the susceptible AO plants. Transcriptome data revealed a total of 21 differentially expressed genes (DEGs) as the core gene set that displayed upregulation in the resistant AK versus susceptible AO after infection with P. asparagi. Kyoto Encyclopedia of Genes and Genomes pathway analysis of these DEGs identified 11 significantly enriched pathways, including flavonoid biosynthesis and primary metabolite metabolism, in addition to plant signaling and defense-related pathways. In addition, comparative single-nucleotide polymorphism and Indel distributions in susceptible AO and resistant AK plants were evaluated using the latest AO reference genome Aspof.V1. The data generated in this study are important resources for advancing Asparagus breeding programs and for investigations of genetic linkage mapping, phylogenetic diversity and plant defense-related genes.

Sex Determination by Two Y-Linked Genes in Garden Asparagus.

The origin and early evolution of sex chromosomes have been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis) is an ideal species to investigate this hypothesis, as the X and Y chromosomes are cytologically homomorphic and evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization directly implicate Y-specific genes that respectively suppress female (pistil) development and are necessary for male (anther) development. Comparison of contiguous X and Y chromosome assemblies shows that hemizygosity underlies the loss of recombination between the genes suppressing female organogenesis (SUPPRESSOR OF FEMALE FUNCTION) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION1 [aspTDF1]). We also experimentally demonstrate the function of aspTDF1. These findings provide direct evidence that sex chromosomes can function through linkage of two sex determination genes.

Asparagus racemosus bZIP transcription factor-regulated squalene epoxidase (ArSQE) promotes germination and abiotic stress tolerance in transgenic tobacco.

A. racemosus is a rich source of pharmacologically active steroidal saponins. Most of the studies are related to its chemistry and pharmacology, but the pathway involved in the biosynthesis of steroidal saponin is not much emphasized. Squalene epoxidase acts as a rate-limiting enzyme in this biosynthesis. In this study, we have selected root specific squalene epoxidase ArSQE from A. racemosus for its characterization. ArSQE was able to complement ergosterol auxotrophy in erg1 yeast mutants. Mutants were sensitive to the antifungal drug terbinafine, whereas ArSQE complementation made them tolerant to the same drug. ArSQE plays a significant role in early germination in transgenic tobacco. The transgenic tobacco seedlings overexpressing ArSQE were tolerant to terbinafine and abiotic stress. Expression analysis of transcripts in ArSQE transgenic lines suggests that it mostly affects ABA, GA, stress, and sterol related functions in transgenic tobacco. Further, root specific MeJA responsive A. racemosus bZIP transcription factors (TFs), ArTGA1 and ArTGA2, were identified that bind to MeJA responsive cis-element present in the promoter region of ArSQE. Characterization of ArSQE of A. racemosus provides new information about its regulation through MeJA responsive bZIP TF along with its role in the development and abiotic stress response in transgenic tobacco.

Agrobacterium tumefaciens-mediated transformation of a hevein-like gene into asparagus leads to stem wilt resistance.

Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 µmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.

Cytogenetic and genomic organization analyses of chloroplast DNA invasions in the nuclear genome of Asparagus officinalis L. provides signatures of evolutionary complexity and informativity in sex chromosome evolution.

BACKGROUND: The transfer of chloroplast DNA into nuclear genome is a common process in plants. These transfers form nuclear integrants of plastid DNAs (NUPTs), which are thought to be driving forces in genome evolution, including sex chromosome evolution. In this study, NUPTs in the genome of a dioecious plant Asparagus officinalis L. were systematically analyzed, in order to investigate the characteristics of NUPTs in the nuclear genome and the relationship between NUPTs and sex chromosome evolution in this species. RESULTS: A total of 3155 NUPT insertions were detected, and they represented approximated 0.06% of the nuclear genome. About 45% of the NUPTs were organized in clusters. These clusters were derived from various evolutionary events. The Y chromosome contained the highest number and largest proportion of NUPTs, suggesting more accumulation of NUPTs on sex chromosomes. NUPTs were distributed widely in all of the chromosomes, and some regions preferred these insertions. The highest density of NUPTs was found in a 47 kb region in the Y chromosome; more than 75% of this region was occupied by NUPTs. Further cytogenetic and sequence alignment analysis revealed that this region was likely the centromeric region of the sex chromosomes. On the other hand, the male-specific region of the Y chromosome (MSY) and the adjacent regions did not have NUPT insertions. CONCLUSIONS: These results indicated that NUPTs were involved in shaping the genome of A. officinalis through complicated process. NUPTs may play important roles in the centromere shaping of the sex chromosomes of A. officinalis, but were not implicated in MSY formation.

Transcriptome analysis of Asparagus officinalis reveals genes involved in the biosynthesis of rutin and protodioscin.

Garden asparagus (Asparagus officinalis L.) is a popular vegetable cultivated worldwide. The secondary metabolites in its shoot are helpful for human health. We analyzed A. officinalis transcriptomes and identified differentially expressed genes (DEGs) involved in the biosynthesis of rutin and protodioscin, which are health-promoting functional compounds, and determined their association with stem color. We sequenced the complete mRNA transcriptome using the Illumina high-throughput sequencing platform in one white, three green, and one purple asparagus cultivars. A gene set was generated by de novo assembly of the transcriptome sequences and annotated using a BLASTx search. To investigate the relationship between the contents of rutin and protodioscin and their gene expression levels, rutin and protodioscin were analyzed using high-performance liquid chromatography. A secondary metabolite analysis using high-performance liquid chromatography showed that the rutin content was higher in green asparagus, while the protodioscin content was higher in white asparagus. We studied the genes associated with the biosynthesis of the rutin and protodioscin. The transcriptomes of the five cultivars generated 336 599 498 high-quality clean reads, which were assembled into 239 873 contigs with an average length of 694 bp, using the Trinity v2.4.0 program. The green and white asparagus cultivars showed 58 932 DEGs. A comparison of rutin and protodioscin biosynthesis genes revealed that 12 of the 57 genes associated with rutin and two of the 50 genes associated with protodioscin showed more than four-fold differences in expression. These DEGs might have caused a variation in the contents of these two metabolites between green and white asparagus. The present study is possibly the first to report transcriptomic gene sets in asparagus. The DEGs putatively involved in rutin and protodioscin biosynthesis might be useful for molecular engineering in asparagus.

An unexpected genetic diversity pattern and a complex demographic history of a rare medicinal herb, Chinese asparagus (Asparagus cochinchinensis) in Korea.

Range-wide population studies of wide spread species are often associated with complex diversity patterns resulting from genetically divergent evolutionary significant units (ESUs). The compound evolutionary history creating such a pattern of diversity can be inferred through molecular analyses. Asparagus cochinchinensis, a medicinally important perennial herb, is in decline due to overharvesting in Korea. Eight A. cochinchinensis populations in Korea and three populations from neighboring countries (China, Japan and Taiwan) were examined using nine nuclear microsatellite loci and three chloroplast microsatellite loci to characterize molecular diversity patterns. The average within-population diversity was limited likely due to long-term bottlenecks observed in all eight populations. High pairwise FST values indicated that the populations have largely diverged, but the divergences were not correlated with geographic distances. Clustering analyses revealed a highly complex spatial structure pattern associated with two ESUs. Approximate Bayesian Computation (ABC) suggests that the two ESUs split about 21,000 BP were independently introduced to Korea approximately 1,800 years ago, and admixed in secondary contact zones. The two ESUs found in our study may have different habitat preferences and growth conditions, implying that the two genetically divergent groups should be considered not only for conservation and management but also for breeding programs in agricultural areas.

Comparative transcriptome analysis of the garden asparagus (Asparagus officinalis L.) reveals the molecular mechanism for growth with arbuscular mycorrhizal fungi under salinity stress.

Soil salinity is one of the most abiotic stress factors that severely affects the growth and development of many plants, which can ultimately threaten crop yield. Arbuscular mycorrhiza fungi (AMF) has been proven to be effective in mitigating salinity stress by symbiosis in many crops. Asparagus officinalis are perennial plants grown in saline-alkaline soil, however, limited information on their molecular mechanisms has restricted efficient application of AMF to garden asparagus under salinity stress. In this study, we conducted a transcriptome analysis on the leaves of garden asparagus to identify gene expression under salinity stress. Seedlings were grown in 4 treatments, including non-inoculated AMF using distilled water (NI), inoculated AMF using distilled water (AMF), non-inoculated with salinity stress (NI + S), and inoculated with salinity stress (AMF + S). A total of 6019 novel genes were obtained based on the reference-guided assembly of the garden asparagus transcriptome. Results revealed that 455 differentially expressed genes (DEGs) were identified when comparing NI + S to AMF + S. However, among the up-regulated DEGs, 41 DEGs were down-regulated, while 242 DEGs had no differences in their expression levels when comparing NI to NI + S. These DEGs' expression patterns may be key induced by AMF under salinity stress. Additionally, the GO and KEGG enrichment analyses of 455 DEGs revealed that these genes mainly participate in the improvement of the internal environment in plant cells, nitrogen metabolic-related processes, and possible photoprotection mechanisms. These findings provide insight into enhanced salinity stress adaptation by AMF inoculation, as well as salt-tolerant candidate genes for further functional analyses.